Thrombin, the most potent activator of platelets, works by activation of the G protein-coupled protease activated receptors PAR1 and PAR4. These receptors initiate G protein signaling cascades leading to increases in intracellular calcium and secretion of autocrine activators, promoting platelet aggregation. Direct thrombin inhibitors such as bivalirudin are rapidly increasing in use in interventional cardiology. However, thrombin's direct role in coagulation, as well as its underappreciated protective role on the vasculature through the thrombin/thrombomodulin activation of Protein C, are blocked by this strategy. An alternate therapeutic target is the PAR receptor system, because selective blockade of thrombin's cellular signaling effects might further decrease bleeding risk. We have preliminary evidence that points to differential signaling through PAR4 compared to PAR1;the molecular signaling process through PAR4 receptors to inside-out integrin signaling, secretion and aggregation will be thoroughly investigated in Aim 1. In Aim 2, we will determine the molecular basis of PAR4 signaling with novel G protein blocking tools designed for studies in human platelets that will inhibit, one at a time, activation of Gq, Gi, Go, G12 or G13 signaling through PAR receptors. Although bivalirudin is increasingly used in acute coronary care, the effect of thrombin inhibition on platelet function has not been thoroughly investigated. Thus we propose two clinical aims to compare platelet PAR signaling and platelet function. In Aim 3, we will investigate patients with stable or unstable coronary artery disease. Their platelet PAR signaling to platelet function will be studied before and during bivalirudin in the Vanderbilt Cardiac Catheterization Laboratory. The second clinical.study aims to evaluate PAR signaling in metabolic syndrome and diabetes, conditions complicated by a high risk of atherothrombotic events. In Aim 4, we have designed a clinical study to address the contribution of PAR receptors to the abnormal platelet activation in these thrombotic states. These detailed mechanistic studies on human platelet PAR1 and PAR4 differential signaling, and studies on effects of bivalirudin on platelet function will provide insight into platelet activation in atherothrombosis, and has the potential to impact future antiplatelet therapeutic strategies.